The objective of this project is to isolate and study the cellular components involved in the interconversion of phylloquinone and phylloquinone-2,3-epoxide as well as the vitamin K-dependent carboxylation of glutamic acid residues in the precursor protein to form gamma-carboxyglutamic acid found in active prothrombin. Emphasis will be placed on purification of phylloquinone epoxidase from the microsomal fraction of rat liver. This enzyme uses the hydroquinone of vitamin K and molecular oxygen to form the 2,3-epoxide. Studies of the purified enzyme will be designed to determine the molecular mechanism by which epoxidation of vitamin K may be coupled to the carboxylation of glutamic acid. This will also require isolation of the vitamin K-dependent carboxylase enzyme from the microsomal fraction. Experiments will also be conducted to study the properties of the proteins carboxylated by vitamin K (i.e., isoelectric focusing) under conditions that result in varying amounts of active prothrombin formation determined alone, and solubilized microsomal protein). The possible role of epoxidation of vitamin K in the carboxylation of protein other than those involved in blood coagulation will also be studied.